MutSβ promotes trinucleotide repeat expansion by recruiting DNA polymerase β to nascent (CAG)n or (CTG)n hairpins for error-prone DNA synthesis

Abstract

Expansion of (CAG)•(CTG) repeats causes a number of familial neurodegenerative disorders. Although the underlying mechanism remains largely unknown, components involved in DNA mismatch repair, particularly mismatch recognition protein MutSβ (a MSH2-MSH3 heterodimer), are implicated in (CAG)•(CTG) repeat expansion. In addition to recognizing small insertion-deletion loop-outs, MutSβ also specifically binds DNA hairpin imperfect heteroduplexes formed within (CAG)n•(CTG)n sequences. However, whether or not and how MutSβ binding triggers expansion of (CAG)•(CTG) repeats remain unknown. We show here that purified recombinant MutSβ physically interacts with DNA polymerase β (Polβ) and stimulates Polβ-catalyzed (CAG)n or (CTG)n hairpin retention. Consistent with these in vitro observations, MutSβ and Polβ interact with each other in vivo, and colocalize at (CAG)•(CTG) repeats during DNA replication. Our data support a model for error-prone processing of (CAG)n or (CTG)n hairpins by MutSβ and Polβ during DNA replication and/or repair: MutSβ recognizes (CAG)n or (CTG)n hairpins formed in the nascent DNA strand, and recruits Polβ to the complex, which then utilizes the hairpin as a primer for extension, leading to (CAG)•(CTG) repeat expansion. This study provides a novel mechanism for trinucleotide repeat expansion in both dividing and non-dividing cells.

Figure 1

MutSβ promotes (CAG)_n or (CTG)_n hairpin retention synthesis in HeLa nuclear extracts. (A) Diagram of hairpin removal/retention assay by Southern blot analysis. The purple bar shows the ³²P-labeled oligonucleotide probe, which specifically anneals to the newly synthesized strand near the BsrBI site. The complete primer sequence of a CTG hairpin substrate used in this study is also shown. (B) Southern blot analysis showing the effect of MutSβ on (CAG)₅ or (CTG)₅ hairpin retention/removal during DNA synthesis in HeLa nuclear extracts. DNA hairpin substrate (0.15 pmol) was incubated with limited amount (30 μg) of HeLa nuclear extracts in the presence of increasing amounts of purified MutSβ. The resulting products were examined by Southern blot analysis. (C, D) Quantification of hairpin-retained products and hairpin-removed products shown in B, respectively. The data were from three independent experiments and the error bar represents SD.

Figure 2

MutSβ stimulates Polβ-induced (CAG)_n and (CTG)_n hairpin retention during DNA synthesis. Unless mentioned otherwise, hairpin retention/removal assays were performed in a 40-μL purified system containing 0.15 pmol (CAG)₅ or (CTG)₅ DNA hairpin substrate, 4 pmol MutSβ, 110 fmol RFC and 2 pmol PCNA in addition to the indicated polymerase (600 fmol Polδ, 260 fmol Polβ). Primer extension products were analyzed by Southern blot analysis as described in Figure 1 legends. (A) Effect of MutSβ on Polβ- and Polδ-catalyzed DNA synthesis using a (CAG)₅ hairpin as primer. (B) Increased hairpin retention activity of Polβ is proportional to the increasing concerntrations of MutSβ. (C) Effect of MutSβ on Polβ- and Polδ-catalyzed DNA synthesis using a (CTG)₅ hairpin as primer. (D) Effect of MutSα:MutSβ ratio on Polβ-catalyzed DNA synthesis using a (CAG)₅ hairpin as primer. Relative hairpin retention activity in each group was calculated by using the hairpin retention activity conducted by Polβ alone as a reference, i.e., dividing hairpin retention level of each reaction with that catalyzed by Polβ alone (see *). The data in A-D were from three independent experiments and the error bar represents SD. (E) Proposed DNA structures for shortened DNA products.

Figure 3

MutSβ-stimulated DNA synthesis by Polβ requires a (CAG)_n or (CTG)_n hairpin. Unless mentioned otherwise, hairpin retention/removal assays were performed in a 40-μL purified system containing 0.15 pmol (CAG)₅ or (CTG)₅ DNA hairpin substrate, 4 pmol MutSβ, 110 fmol RFC, 2 pmol PCNA and 260 fmol Polβ. DNA synthesis products were analyzed by Southern blot analysis as described in Figure 1 legends. (A) Comparison of hairpin retention activity of Polβ in reactions with or without MutSβ. DNA substrate in non-hairpin reactions (reactions 1 and 2) used a ssM13mp18 derivative containing 15 CAG repeats to match the size of hairpin-retained products. (B) Hairpin retention activity in reaction with heat-inactivated MutSβ. (C) Dependence of hairpin retention activity on the incubation order of MutSβ and Polβ. Relative hairpin retention activity in each group was calculated by using the hairpin retention activity conducted by Polβ alone as a reference, i.e., dividing hairpin retention activity of each reaction with that catalyzed by Polβ alone (see *). The data were from three independent experiments and the error bar represents SD.

Figure 4

MutSβ physically interacts with Polβ. (A) Gel shift assay to determine the interaction of MutSβ, Polβ and a ³²P-labeled primer extension substrate containing a (CAG)₅ hairpin. (B, C) Co-immunoprecipitation (Co-IP) assay to determine interactions between MutSβ and Polβ in HeLa nuclear extract. Co-IP by IgG was used as a negative control. (D) Far-western blot analysis showing direct interaction between purified MutSβ and Polβ. PCNA and polδ were used as positive and negative controls, respectively.

Figure 5

MutSβ-Polβ complex colocalizes with (CAG)•(CTG) repeats during DNA synthesis. (A) Confocal immunofluorescence analysis showing MSH3 and Polβ foci and their colocalization in HeLa cells in G1 phase (top) and S phase (bottom). (B) DNA sequence of 5 end Cy3-labeled oligonucleotide used in FISH analysis and its mapping site at the ectopic c-Myc replication origin in HeLa-(CAG)₄₅ cells. (C, D) Confocal immunofluorescence analysis showing MSH3-(CTG)₄₅ and Polβ-(CTG)₄₅ colocalizations in HeLa-(CAG)₄₅ cells in S phase, respectively. (E) Confocal immunofluorescence analysis showing colocalization of MSH3, Polβ and (CTG)₄₅ in HeLa-(CAG)₄₅ cells in S phase (bottom), with HeLa cells without the ectopic (CAG)₄₅ sequence as a negative control (top). β and 3 represent Polβ and MSH3, respectively. (F) Percentage of cells showing colocalization of MSH3, Polβ and (CTG)₄₅ repeats.

Figure 6

Proposed model for MutSβ's role in promoting (CAG)•(CTG) repeat expansion. During DNA synthesis (i.e., DNA replication or DNA repair), (CAG)•(CTG) repeats form a hairpin via strand slippage. The hairpin can be removed by Polδ (I). However, hairpin binding by MutSβ recruits Polβ to the complex, where Polβ uses the hairpin as a primer to add several nucleotides to the 3′ end of hairpin. The resulting hairpin product can be utilized for the high-fidelity and highly processive DNA synthesis by Polδ, leading to hairpin retention (II). Alternatively, Polβ can carry out the hairpin retention DNA synthesis independent of Polδ (III).